Lower extremity peripheral arterial occlusive disease poses a unique challenge to traditional endovascular therapies. The diffuse nature of lower extremity atherosclerotic disease, the presence of chronic total occlusions, poor distal runoff, and the presence of limb ischemia have contributed to disappointing results in treatment. Peripheral arterial disease (PAD) affects ˜12 million people in the United States and approximately 220,000 to 240,000 amputations occur yearly in the US and Europe. PAD encompasses chronic limb ischemia, which progresses into critical limb ischemia leading to the distal limb at risk of amputation, and acute limb ischemia, with a rapid loss of blood flow damaging tissue within hours. Critical Limb ischemia (CLI) is often associated with diabetes, resulting in compromised vasculature and exaggerated tissue damage. A separate chronic condition, Buerger's Disease, compromises blood flow to the hands and feet resulting in the loss of fingers and toes. Chronic PAD generally results in poor wound healing, ulcers and tissue necrosis in limbs and extremities that may result in loss of the affected limb as a result of non-traumatic amputation.
In CLI, patients present with chronic ischemic rest pain, presence of tissue ulcers, or gangrene as a result of reduced blood flow due to proven occlusive disease. The condition is the progression of PAD as diagnosed by a low or a lack of pulse in the foot, low ankle brachial index (ABI, blood pressure in ankle/blood pressure in arm <0.6) reduced blood pressure in toe (<30-50 mm Hg), reduced transcutaneous oxygen (<30-50 mm Hg). Occlusion of arteries is usually caused by atherosclerosis within large communicating arteries that is confirmed using angiography or duplex ultrasound scanning. Once diagnosed with critical limb ischemia, patients have a risk of limb loss of 10 to 40% over the first year. CLI patients are usually also at risk for myocardial infarction, stroke and coronary artery disease.
PAD is a common cardiovascular complication in patients with diabetes. In contrast to peripheral arterial disease in non-diabetic individuals, it is more prevalent and, because of the distal territory of vessel involvement and its association with peripheral neuropathy, it is more commonly asymptomatic. It is estimated that approximately 20% of patients with symptomatic PAD are diabetic. Diabetics with limb ischemia are at a greater risk of tissue damage. The pain felt by non-diabetics while walking, ie the first indication of limb ischemia, may not be perceived by diabetics due to the presence of peripheral neuropathy. Diabetic patients may note pain in the thighs and buttocks while walking due to the loss of reserve blood flow or have a sensation of the muscles being tired.
Patients with mild cases of limb ischemia are treated medically with antiplatelet (clopidogrel) and vasodilator therapy (pentoxifylline). Risk modification is also used to eliminate smoking and control hypertension, which are contributing factors to PAD. Monitored exercise regimens are the most effective treatment of PAD with sessions lasting longer than 30 minutes at least three times per week, with programs lasting at least six months (Gey et al 2004).
Patients with moderate limb ischemia, moderate to severe claudication and walking distances of 200 meters or less are candidates for percutaneous transluminal angioplasty along with medical therapy (Gray et al 2008). Patients with severe symptoms including ischemic rest pain and chronic ulcers are candidates for angioplasty with and without stent insertion and/or bypass surgery. Unfortunately, surgical procedures that restore blood flow commonly result in the loss of patency of the vessels over one to two years with up to 20% of patients having re-stenosis of the affected arteries. Complications also arise in up to 25% of patients having poor healing at the wound site.
Approximately 20 to 30% of patients with the most severe symptoms of CLI are not considered candidates for vascular or endovascular procedures and are, therefore, considered for amputation. Patients with necrotic tissue and wet or dry gangrene are normally considered candidates for amputation, although some cases of dry amputation resolve with the affected tissue being shed. Amputation is also considered in cases of advanced ischemia associated with a low ankle brachial index value (<0.3).
Acute limb ischemia (ALI) is defined as any sudden decrease or worsening in limb perfusion (Gray et al 2008). The causes of ALI are usually an acute thrombotic occlusion of a pre-existing stenotic arterial segment (60% of cases) or an embolus (clot) usually originating from the heart or from a segment of an atherosclerotic plaque (30% of cases) stopping blood flow in a peripheral artery. Blockages also occur from trauma, fractures, blunt and penetrating injuries and from complications of surgical procedures. Damage from ALI occurs in the affected muscles within hours of onset.
Patients with ALI present with a generalised set of symptoms resulting from loss of blood flow to the limb. These symptoms include; Pain, Pallor (whiteness in the limb), Pulseless extremity, Poikolothermia (limb assumes surrounding temperature), Parasthesia (tingling sensation) and Paralysis. As the period of ALI lengthens the limb starts to have a mottled appearance as blood begins to coagulate. The presence of anesthesia, with the patient unable to feel touch in the limb, and being unable to wiggle the toes or fingers are the key to diagnosing complete ischemia, requiring emergency surgical treatment (Callum & Bradbury 2000).
Prompt treatment of ALI is necessary as a few hours of resolution can make the difference between limb amputation (and possible death) and recovery of limb function. Heparin administration is usually the first line of treatment to limit propagation of a thrombus and protect the collateral circulation of the affected limb (Gray et al 2008). Patients with limbs that are viable or with a marginal threat (no or minimal sensory loss and audible venous signals) are usually treated with catheter-directed thrombolytic therapy. Cases with an immediate threat of damage (pain, muscle weakness and inaudible arterial signals) may be treated using thrombolytic therapy, a percutaneous thrombectomy device or surgical bypass. Patients presenting with non-viable limbs having irreversible damage (loss of sensation, paralysis with muscle rigor and inaudible arterial and venous signals) face amputation of the limb.
Patients with ALI still face risk of secondary damage to the limb after restoration of blood flow due to reperfusion injury. Tissue suffers from peroxidation and neutrophil invasion along with swelling of the limb. Peripheral nerve injury suffered from the ischemic period and resulting reperfusion may lead to chronic pain. Stress in the peripheral organs from products in the blood as a result of the ischemic episode can lead to damage and organ failure. Patients suffering from coronary artery disease have increased risk of death after ALI.
There are approximately 200,000 cases of ALI in the US every year. Of those cases 10 to 20% of cases have in-hospital mortality due to heart failure and or recurrent embolism.
There is also an angiogenic response to ischemia. Ischemia-induced angiogenesis involves the growth of new microvasculature and capillary beds. Cells within the ischemic tissue respond to hypoxia (low oxygen levels) and induce angiogenesis through the hypoxia-inducible factor 1 (HIF-1).
Disease states related to reductions in vascular perfusion may be treated using strategies that promote re-vascularisation of tissue. Most approaches for promotion of vascularisation have centred on delivery of a gene or growth factor, typically VEGF and/or fibroblast growth factor (FGF). Although clinical trials for angiogenic therapy have been shown to alleviate secondary symptoms, they have failed to demonstrate improvements in exercise performance, the US FDA primary endpoint for approval of an angiogenic agent. Some side-effects have been associated with the administration of VEGF and FGF including hypotension, edema, renal insufficiency and vascular leakage.
Angiogenesis stimulated by VEGF exposure does not consistently form stable functional vasculature. Long term exposure to VEGF is required to produce stable microvasculature that does not degrade after withdrawal of the VEGF stimulus. Concentrations of VEGF are also critical as low dosages of VEGF result in vessels with increased permeability leading to oedema and high doses of VEGF can result in formation of hemangioma and vascular leakage.
Implantation of dissolvable matrices embedded with growth factors may be used as a source for growth factors. Growth factors (FGF and VEGF) may be incorporated into a biodegradable scaffolds (for example, PLGA based) that is slowly released as the matrix degrades. Such an approach allows a timed release of growth factors delivered locally to the site of interest. Once the new vasculature is established, growth factors may be administered in a controlled fashion to maintain optimal functionality of the new vessels.
Cell-based therapies have also been proposed. Endothelial progenitor cells (EPC) originating in the bone marrow play a significant role in endogenous neovascularisation of injured vessels. EPC transplantation has been shown to induce new vessel formation in ischemic myocardium and hind limb, and to accelerate re-endothelialisation of injured vessels and prosthetic vascular grafts in humans and in various animals models. EPCs have been demonstrated as a potential therapy for a cell based strategy for the rescue and repair of ischemic tissues and injured blood vessels. EPCs are thought to originate from a multiple precursors in the bone marrow. The cells have a high proliferative potential albeit with a finite number of cell divisions. Numbers of EPCs are low under normal conditions, however, stimulation with exogenous cytokines and hormone raises the number of EPCs several fold. The mechanisms governing the mobilisation, homing and differentiation of EPCs in vivo remain largely unknown. EPCs can be isolated from peripheral blood and expanded to provide sufficient numbers for autologous transplantation.
Numbers of EPCs are reduced in patients with significant risk of cardiovascular disease. In patients with severe coronary artery disease, the potency of EPCs as defined by colony-forming capacity and migratory activity was markedly reduced and associated with reduced neovascularisation in hind limb ischemia (Heeschen et al 2004). Similarly, numbers of EPCs are reduced in patients with type I or type II diabetes. The ability of EPCs to induce angiogenesis in vitro is also reduced. The reduction of the numbers of EPCs and of their activity suggest that their deficits are involved with some of the vascular complications associated with diabetes, such as endothelial dysfunction, that predispose patients to diffuse atherosclerosis and impaired neovascularisation after ischemic events.
The implantation of EPC progenitor cells into the ischemic limb has been shown to promote new vessel formation (neoangiogenesis) in the affected limb. Administration of bone marrow cells into a mouse model of hind-limb ischemia was the first observation that cell therapy can contribute to neoangiogenesis following an ischemic insult (Asahara et al 1999). In 2002, Tateishi-Yuyama et al. first reported that autologous bone marrow mononuclear cell implantation was an effective treatment for patients with ischemic limbs due to peripheral arterial disease (Tateishi-Yuyama et al 2002). In additional studies, the optimal dose of autologous bone marrow derived cells for the treatment of limb ischemia was reported to be no less than 1×105 cells, and the optimal implantation dose was 1×108 cells (Gu et al 2006; Liao & Zhao 2008).
Functional studies monitoring treadmill running in rats with hind limb ischemia has shown increases in both perfusion of the limb and in performance of treated animals. Bone marrow administration has also increased angiogenesis in diabetic rats with hind limb ischemia.
A number of variations of treatment with bone marrow cell populations into the muscle of ischemic hind limb have demonstrated a promotion of angiogenesis via the generation of angiogenic factors and by promotion of endothelial progenitor cells.
The feasibility of cell implantation using autologous bone marrow derived cells including hematopoietic, endothelial and mesenchymal stem cells or their combinations has been demonstrated in a number of clinical studies, with varying levels of benefit being observed. While overall positive results have been observed, the limitations are clear: for older and diabetic patients, the therapeutic potential of the patients' own cells is diminished. In addition, the inconvenience and cost of extraction, isolation, purification, release testing and readministration of patient specific stem cells is a hindrance to widespread reimbursable medical application.
Real clinical and industrial progress in cell implantation for the treatment of peripheral ischemia in all its manifestations requires a stem cell product which is able to meet all the desired characteristics:                High therapeutic effectiveness        Excellent safety profile        Standardised for all patients (i.e., not patient specific)        Available on demand, even in emergency situations.        Low cost of goods (compared to patient-specific treatments)        
There is therefore a clear need for improved treatments for peripheral arterial disease, including limb ischemia.